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Background: According to WHO (CISMAC. Centre for Intervention Science in Maternal and Child health), the antimicrobial resistant bacteria considered to be clinically most important for human health and earmarked for surveillance include extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, carbapenem-resistant bacteria, methicillin-resistant (MRSA) and, macrolide-lincosamide-streptogramin B -resistant vancomycin-resistant (VRSA) Staphylococcus aureus and vancomycin-resistant Enterococcus (VRE). If these bacteria are carried in the female genital tract, they may be transmitted to the neonate causing local or systemic neonatal infections that can be difficult to treat with conventionally available antimicrobials. In order to develop effective treatment strategies, there is need for updated information about the prevalence of colonization with important antimicrobial-resistant pathogens. Objective: We sought to estimate the prevalence of vaginal colonization with potentially pathogenic and clinically important AMR bacteria among women in labour in Uganda and to identify factors associated with colonization. Methods: We conducted a cross-sectional study among HIV-1 and HIV-2 negative women in labour at three primary health care facilities in Uganda. Drug susceptibility testing was done using the disk diffusion method on bacterial isolates cultured from vaginal swabs. We calculated the prevalence of colonization with potentially pathogenic and clinically important AMR bacteria, in addition to multidrug-resistant (MDR) bacteria, defined as bacteria resistant to antibiotics from ≥ 3 antibiotic classes. Results: We found that 57 of the 1472 enrolled women (3.9% prevalence; 95% Confidence interval [CI] 3.0%, 5.1%) were colonized with ESBL-producing Enterobacteriaceace, 27 (1.8%; 95% CI 1.2%, 2.6%) were colonized with carbapenem-resistant Enterobacteriaceae, and 85 (5.8%; 95% CI 4.6%, 7.1%) were colonized with MRSA. The prevalence of colonization with MDR bacteria was high (750/1472; 50.9%; 95% CI 48.4%, 53.5%). Women who were ≥ 30 years of age had higher odds of being colonized with MDR bacteria compared to women aged 20–24 years (OR 1.6; 95% CI 1.1, 2.2). Conclusion: Most of the women included in our study were vaginally colonized with potentially pathogenic MDR and other clinically important AMR bacteria. The high prevalence of colonization with these bacteria is likely to further increase the incidence of difficult-to-treat neonatal sepsis.
We conducted a cross-sectional study between July 2016 and July 2018 at three primary health care facilities in and close to Kampala in central Uganda: Mukono General Hospital (formerly Mukono Health Centre IV), Kawaala Health Centre III, and Kitebi Health Centre III [15]. This study was nested within the Chlorhexidine Trial, which is a randomized controlled assessing whether a single application of 4% chlorhexidine solution on the umbilical cord stump immediately after birth reduces the risk of omphalitis and severe illness [16]. Of the 1658 women in labour who were screened for this study, we recruited 1472 who had already agreed to being enrolled in the above-mentioned Chlorhexidine Trial. The inclusion criteria for the trial were: mother was negative for HIV-1 and HIV-2 and gave birth on a weekday, the newborn weighed > 1.5 kgs, had no severe congenital anomalies, had no obvious signs of umbilical cord stump infection and had no severe illness on the day it was born. We enThe sample size was calculated for the trial but not for the present study. With this sample size we would obtain a very high (0.7% to 2.6%) absolute precision, i.e. the difference between the upper limit and the lower limit of the 95% confidence interval (CI) for prevalence estimates ranging from 2 to 50%. Midwives obtained oral consent to collect a specimen from women in labour. After birth, a study nurse confirmed the verbal consent by obtaining written consent. In the subsequent interview, study nurses collected socio-demographic and clinical information from study participants using Open Data Kit-based standardized questionnaires [17]. Trained midwives collected the vaginal swabs during labour. A Regular Rayon sterile swab (Copan Diagnostics Inc., Murrieta, CA) was carefully inserted halfway between the introitus and cervix, avoiding contamination from the cervical mucus. The swab was then gently pressed against the vaginal wall, rotated to collect the specimen, and then removed, carefully avoiding contact with other parts of the body. The swabs were immediately stored in Amies Agar Gel without Charcoal transport medium (Copan Diagnostics Inc.) and transported daily in a cold box holding a temperature of 10–25 °C to MBN Clinical Laboratories Ltd in Kampala, which is a private research and diagnostic laboratory currently undergoing accreditation, where the swabs were immediately processed. We did not culture the specimens anaerobically because of the added cost and effort and because anaerobic bacteremia is uncommon in neonates [2]. The specimens were streaked onto blood agar containing 5% in-house produced sheep blood and onto MacConkey agar (both Biolab Inc., Budapest, Hungary) and incubated aerobically at 35–37 °C for 18–24 h for isolation of single colonies. The blood agar plates were further incubated for a total of 72 h to enable isolation of slow-growing colonies. From each of the two plates, one representative of each morphologically distinct colony was picked and streaked onto new plates and 1–5 resulting colonies from each of them were pooled in saline solution and subjected to further species determination and classification as described below, before being stored at −80 °C in Brain Heart Infusion broth with 20% glycerol. Identification of bacterial species was mainly done based on colony morphology, Gram staining and on standard biochemical tests. S. aureus was identified with the catalase, slide coagulase, mannitol fermentation, and DNase tests. The bile esculin test was performed to identify Enterococcus spp. Putative streptococcal isolates were grouped into different Lancefield groups with the Streptococcal Grouping Kit (Oxoid Ltd., Basingstoke, Hants, UK). Lactose and non-lactose fermenting colonies of gram-negative bacilli were identified based on morphology on MacConkey agar, and the isolates were characterized on the species level by performing standard biochemical tests such as Sulphide Indole Motility (SIM test), gas production, citrate test, urease test and oxidase test [18]. If two isolates from the same specimen were of the same species but had different biochemical characteristics, we included both isolates in the analyses. We considered gram-negative isolates representing E. coli, K. pneumoniae, Citrobacter spp., Enterobacter spp., Acinetobacter spp., K. oxytoca, Pseudomonas spp., and Proteus spp. and gram-positive isolates representing S. aureus, Enterococcus spp., Group A Streptococcus, and Group B Streptococcus to be potentially pathogenic bacteria and they were thereby included in the present study. Antimicrobial drug susceptibility testing of the bacterial isolates was performed using the disk diffusion method as described in the 2017 Performance Standards published by the Clinical Laboratory Standard Institute (CLSI) [19]. We also tested for the antibiotics recommended by the same standard. For gram-positive isolates, we tested against the following antibiotic resistance discs purchased from Biolab Inc.: penicillin (10 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), chloramphenicol (30 µg), tetracycline (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), erythromycin (15 µg), oxacillin (1 µg), vancomycin (30 µg), ceftriaxone (30 µg), and linezolid (30 µg). For Gram-negative isolates, we tested against the following discs: trimethoprim-sulfamethoxazole (1.25/23.75 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), gentamicin (10 µg), amikacin (10 µg), ampicillin (10 µg), amoxicillin-clavulanic acid (20/10 µg), ceftriaxone (30 µg), cefuroxime (30 µg), ceftazidime (30 µg), tetracycline (30 µg), piperacillin (100 µg), piperacillin-tazobactam (100/10 µg), colistin (10 µg), and imipenem (10 µg). The inhibition zone diameters were measured after incubation at 35–37 °C for 24 h, and we considered an isolate to be resistant (i.e. non-susceptible) if the measurements indicated resistance or intermediate resistance to the given drug. To identify ESBL-producing Enterobacteriaceae, we used the combination disk method [20] where a combination disk containing 30 µg ceftazidime and 10 µg clavulanic acid was placed 15 mm from a 30 µg ceftazidime disk on a Mueller–Hinton agar plate. Isolates that had clear zones that were ≥ 5.0 mm larger around the combination disk than around the ceftazidime disk were considered to represent ESBL-producing bacteria. We considered isolates that were resistant to imipenem to be carbapenem-resistant based on CLSI guidelines. To identify methicillin-resistant S. aureus (MRSA) genotypically, we performed multiplex PCR-based identification of MRSA of most S. aureus isolates, as described by McClure et al. [21]. In this assay, the presence of the mecA methicillin resistance gene was used to identify MRSA, while the presence of the gene for the Panton-Valentine Leukocidin (PVL) virulence factor was a marker for community-acquired MRSA [22]. The completed reaction was separated on a 2% agarose gel stained with ethidium bromide, and the amplicons were visualized by using a UV trans-illuminator. To identify S. aureus isolates that had the macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype, we performed the D-test [23]. In this test, disks with 15 µg erythromycin and with 2 µg clindamycin were placed 15 mm apart. If the isolate was resistant to both erythromycin and clindamycin, the isolate was considered to have a constitutive MLSB resistance phenotype (cMLSB), while if it was resistant to erythromycin and susceptible to clindamycin, but there was a D-shaped inhibition zone around the clindamycin disk, we considered the isolate to have an inducible MLSB resistance phenotype (iMLSB). We considered Enterococcus spp. and S. aureus isolates that are resistant to vancomycin to represent VRE and VRSA, respectively. We used the definition proposed by Magiorakos et al. [9], i.e. that isolates non-susceptible to ≥ 1 antibiotic in ≥ 3 of the antibiotic classes were considered MDR. The antibiotic selection was based on the 2017 Performance Standards from CLSI [19], which differs slightly from other commonly used standards, such as those published by EUCAST [24]. The antibiotic classes and antibiotics (given in parentheses) used for the MDR definition included penicillins (ampicillin, piperacillin, penicillin), penicillins and beta-lactamase inhibitors (amoxicillin-clavulanic acid), antipseudomonal penicillins and beta-lactamase inhibitors (piperacillin-tazobactam), non-extended-spectrum beta-lactams such as second generation cephalosporins (cefuroxime), extended spectrum beta lactams such as third generation cephalosporins (ceftriaxone, ceftazidime), carbapenems (imipenem), fluoroquinolones (ciprofloxacin), phenicols (chloramphenicol), folate pathway inhibitors (Trimethoprim-sulfamethoxazole), aminoglycoside (gentamicin, amikacin), anti-staphylococcal beta lactams (oxacillin), glycopeptides (vancomycin), macrolides (erythromycin), tetracyclines (tetracycline), and oxazolidinones (linezolid). As seen in Tables Tables11 and and2,2, we did not test for resistance for a given antibiotic if the species is known to be naturally resistant to the antibiotic. Vaginal colonization with antimicrobial drug-resistant potentially pathogenic gram-negative bacteria among study women in labor aNA indicates that the antibiotic was not tested or was not relevant for the given organism bMDR excluding ESBL-producing and carbapenem-resistant bacteria cThe two numbers in parentheses indicate number of colonized women and the number of isolates of each given species, respectively Vaginal colonization with antimicrobial drug-resistant potentially pathogenic gram-positive bacteria among study women in labor aNA indicates that the information is not relevant for the given resistance pattern bMDR excluding MRSA, VRSA, VRE, iMLSB- and cMLSB-resistant S. aureus cThe two numbers in parentheses indicate number of colonized women and the number of isolates of each given species, respectively To identify potential risk factors for colonization with different AMR bacteria in the logistic regression models described below, we included the following exposure information, which were acquired during interviews with the mothers after they had given birth. PROM defined as rupture of membranes before the start of labour [25], parity, maternal level of education, maternal age, hospitalization during pregnancy, antenatal care attendance, ownership of domestic animals at home and socioeconomic status. As a measure of socioeconomic status, we used principal component analysis on an asset index that we generated by evaluating the woman’s access to or ownership of cupboards, radios, televisions, a mobile phone, refrigerator, motorcycle, car, ownership of a house, and presence of cemented walls, flushing toilet, and having three or more rooms in the house. Socioeconomic status was divided into 5 levels, where the poorest women were categorized as belonging to the 1st quintile while the richest were categorized as belonging in the 5th quintile. The statistical analyses were done using Stata version 15.0 (StataCorp, College Station, Texas, USA). We obtained the overall prevalence of MDR colonization by dividing the number of women colonized with MDR bacteria by the total number of women enrolled into the study. To obtain the overall prevalence of resistance with clinically important bacteria such as MRSA, MLSB-resistant S. aureus, VRSA, VRE, ESBL and carbapenem-resistant bacteria, we divided the number of women colonized with such bacteria by the total number of enrolled women in the study. All proportions were reported with their respective 95% confidence intervals, which were calculated using the exact method. We performed multivariable logistic regression analyses to explore the association between selected exposures (maternal age, maternal education, socioeconomic status, gravidity, number of antenatal visits, hospitalization during pregnancy, ownership of domestic animals) and our primary outcome of vaginal colonization with MDR bacteria and secondary outcomes including ESBL and MRSA bacteria. We chose to conduct exploratory multivariable analyses because the exposures selected potentially confounded one another. We used the estat vif command in STATA to test the models for potential multicollinearity between the independent variables, as indicated by one or more variance inflation factor estimates being > 10. None of our models appeared to have potential multicollinearity issues.
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